Including Insulin-like Growth Factor I Research Articles

A role for astrocytic insulin-like growth factor I receptors in the response to ischemic insult

Increased neurotrophic support, including insulin-like growth factor I (IGF-I), is an important aspect of the adaptive response to ischemic insult. However, recent findings indicate that the IGF-I receptor (IGF-IR) in neurons plays a detrimental role in the response to stroke. Thus, we investigated the role of astrocytic IGF-IR on ischemic insults using tamoxifen-regulated Cre deletion of IGF-IR in glial fibrillary acidic protein (GFAP) astrocytes, a major cellular component in the response to injury. Ablation of IGF-IR in astrocytes (GFAP-IGF-IR KO mice) resulted in larger ischemic lesions, greater blood-brain-barrier disruption and more deteriorated sensorimotor coordination. RNAseq detected increases in inflammatory, cell adhesion and angiogenic pathways, while the expression of various classical biomarkers of response to ischemic lesion were significantly increased at the lesion site compared to control littermates. While serum IGF-I levels after injury were decreased in both control and GFAP-IR KO mice, brain IGF-I mRNA expression show larger increases in the latter. Further, greater damage was also accompanied by altered glial reactivity as reflected by changes in the morphology of GFAP astrocytes, and relative abundance of ionized calcium binding adaptor molecule 1 (Iba 1) microglia. These results suggest a protective role for astrocytic IGF-IR in the response to ischemic injury.

All-cause mortality in patients with acromegaly treated with pegvisomant: an ACROSTUDY analysis.

To examine all-cause mortality rates in patients with acromegaly on pegvisomant and identify pertinent risk factors, including insulin-like growth factor I (IGF-I). Retrospective cohort analysis of data from ACROSTUDY (global surveillance study of patients with acromegaly treated with pegvisomant). Kaplan-Meier analyses and Cox regression techniques were used to examine survival rates. Standardized mortality ratios (SMR) with reference to general population (WHO GBD 2016) were estimated. Multiplicative multiple Poisson regression models were used to characterize the association between SMR, IGF-I, and other risk factors associated with mortality risk. The study consisted of 2077 subjects who were followed for a median interval of 4.1 years, contributing to 8957 patient-years. Higher on-treatment IGF-I (P = 0.0035), older attained age (P < 0.0001), and longer duration of acromegaly (>10 years) before starting pegvisomant (P = 0.05) were associated with higher mortality rates. In reference to general population rates, higher SMR (1.10, 1.42, and 2.62, at attained age 55 years) were observed with higher serum IGF-I category (SMR trend: 1.44 (44%)/per fold level of IGF-I/ULN (95% CI: 1.10, 1.87), P = 0.0075). SMR increased per year of younger attained age (1.04 (1.02-1.04), P < 0.0001) and were higher for longer disease duration (>10 years) before starting pegvisomant (1.57 (1.02, 2.43), P = 0.042). Serum IGF-I levels within the normal range during pegvisomant therapy were associated with all-cause mortality rates that were indistinguishable from the general population. Higher on-treatment IGF-I, older attained age, and longer duration of acromegaly before starting pegvisomant are associated with higher all-cause mortality rates. Younger patients with uncontrolled acromegaly have higher excess all-cause mortality rates in comparison with older patients.

Growth Hormone Stimulation Tests in Children with Kabuki Syndrome

Background/Aims: Kabuki syndrome is a multiple congenital malformation syndrome with a variety of clinical features including short stature. The cause of this postnatal short stature remains unknown. Methods: Eighteen children with genetically proven Kabuki syndrome (8 boys and 10 girls; ages 3.3-9.9 years, with a mean of 6.7 years) who underwent growth hormone (GH) stimulation tests were evaluated in a prospective study. Two GH stimulation tests were conducted, including insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) serum levels. GH stimulation peaks in relation to age, sex, height, body mass index (BMI), IGF-I, and IGFBP-3 SD scores (SDS) were analyzed. Results: Five of the 18 children (27.8%) were biochemically GH deficient. This was not correlated with BMI SDS. Of all patients, only 1 had an IGF-I below -2 SD and did not fulfill the GH deficiency criteria. The mean IGF-I level was below normal (-0.8 SD). All subjects had normal IGFBP-3 levels. Conclusions: The utility of performing GH stimulation tests on Kabuki syndrome children as an indication of GH status in short stature is questionable. IGF-I levels did correlate neither with the GH stimulation peak nor consequently with the diagnosis of GH deficiency.

Chronic lung injury in the neonatal rat: Up-regulation of TGFβ1 and nitration of IGF-R1 by peroxynitrite as likely contributors to impaired alveologenesis

Postnatal alveolarization is regulated by a number of growth factors, including insulin-like growth factor-I (IGF-I) acting through the insulin-like growth factor receptor-1 (IGF-R1). Exposure of the neonatal rat lung to 60% O2 for 14 days results in impairments of lung cell proliferation, secondary crest formation, and alveologenesis. This lung injury is mediated by peroxynitrite and is prevented by treatment with a peroxynitrite decomposition catalyst. We hypothesized that one of the mechanisms by which peroxynitrite induces lung injury in 60% O2 is through nitration and inactivation of critical growth factors or their receptors. Increased nitration of both IGF-I and IGF-R1 was evident in 60% O2-exposed lungs, which was reversible by concurrent treatment with a peroxynitrite decomposition catalyst. Increased nitration of the IGF-R1 was associated with its reduced activation, as assessed by IGF-R1 phosphotyrosine content. IGF-I displacement binding plots were conducted in vitro using rat fetal lung distal epithelial cells which respond to IGF-I by an increase in DNA synthesis. When IGF-I was nitrated to a degree similar to that observed in vivo there was minimal, if any, effect on IGF-I displacement binding. In contrast, nitrating cell IGF-R1 to a similar degree to that observed in vivo completely prevented specific binding of IGF-I to the IGF-R1, and attenuated an IGF-I-mediated increase in DNA synthesis. Additionally, we hypothesized that peroxynitrite also impairs alveologenesis by being an upstream regulator of the growth inhibitor, TGFβ1. That 60% O2-induced impairment of alveologenesis was mediated in part by TGFβ1 was confirmed by demonstrating an improvement in secondary crest formation when 60% O2-exposed pups received concurrent treatment with the TGFß1 activin receptor-like kinase, SB 431542. That the increased TGFβ1 content in lungs of pups exposed to 60% O2 was regulated by peroxynitrite was confirmed by its attenuation by concurrent treatment with a peroxynitrite decomposition catalyst. We conclude that peroxynitrite contributes to the impaired alveologenesis observed following the exposure of neonatal rats to 60% O2 both by preventing binding of IGF-I to the IGF-R1, secondary to nitration of the IGF-R1, and by causing an up-regulation of the growth inhibitor, TGFβ1.

Lipid rafts as major platforms for signaling regulation in cancer

Cell signaling does not apparently occur randomly over the cell surface, but it seems to be integrated very often into cholesterol-rich membrane domains, termed lipid rafts. Membrane lipid rafts are highly ordered membrane domains that are enriched in cholesterol, sphingolipids and gangliosides, and behave as major modulators of membrane geometry, lateral movement of molecules, traffic and signal transduction. Because the lipid and protein composition of membrane rafts differs from that of the surrounding membrane, they provide an additional level of compartmentalization, serving as sorting platforms and hubs for signal transduction proteins. A wide number of signal transduction processes related to cell adhesion, migration, as well as to cell survival and proliferation, which play major roles in cancer development and progression, are dependent on lipid rafts. Despite lipid rafts harbor mainly critical survival signaling pathways, including insulin-like growth factor I (IGF-I)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling, recent evidence suggests that these membrane domains can also house death receptor-mediated apoptotic signaling. Recruitment of this death receptor signaling pathway in membrane rafts can be pharmacologically modulated, thus opening up the possibility to regulate cell demise with a therapeutic use. The synthetic ether phospholipid edelfosine shows a high affinity for cholesterol and accumulates in lipid rafts in a number of malignant hematological cells, leading to an efficient invitro and invivo antitumor activity by inducing translocation of death receptors and downstream signaling molecules to these membrane domains. Additional antitumor drugs have also been shown to act, at least in part, by recruiting death receptors in lipid rafts. The partition of death receptors together with downstream apoptotic signaling molecules in membrane rafts has led us to postulate the concept of a special liquid-ordered membrane platform coined as "cluster of apoptotic signaling molecule-enriched rafts" (CASMER), referring to raft platforms enriched in apoptotic molecules. CASMERs act as scaffolds for apoptosis signaling compartmentalization, facilitating and stabilizing protein-protein interactions by local assembly of cross-interacting molecules, which leads to apoptosis amplification and a decrease in apoptotic signal threshold. Edelfosine also displaced survival PI3K/Akt signaling from lipid rafts, leading to Akt inhibition, in mantle cell lymphoma cells. Thus, membrane rafts could act as scaffold structures where segregation of pro- from anti-apoptotic molecules could take place. In this review, we summarize our view of how reorganization of the protein composition of lipid raft membrane domains regulates cell death and therefore it might be envisaged as a novel target in the treatment of cancer.

Stem cell conditioned medium improves acute lung injury in mice: in vivo evidence for stem cell paracrine action

Mortality and morbidity of acute lung injury and acute respiratory distress syndrome remain high because of the lack of pharmacological therapies to prevent injury or promote repair. Mesenchymal stem cells (MSCs) prevent lung injury in various experimental models, despite a low proportion of donor-derived cell engraftment, suggesting that MSCs exert their beneficial effects via paracrine mechanisms. We hypothesized that soluble factors secreted by MSCs promote the resolution of lung injury in part by modulating alveolar macrophage (AM) function. We tested the therapeutic effect of MSC-derived conditioned medium (CdM) compared with whole MSCs, lung fibroblasts, and fibroblast-CdM. Intratracheal MSCs and MSC-CdM significantly attenuated lipopolysaccharide (LPS)-induced lung neutrophil influx, lung edema, and lung injury as assessed by an established lung injury score. MSC-CdM increased arginase-1 activity and Ym1 expression in LPS-exposed AMs. In vivo, AMs from LPS-MSC and LPS-MSC CdM lungs had enhanced expression of Ym1 and decreased expression of inducible nitric oxide synthase compared with untreated LPS mice. This suggests that MSC-CdM promotes alternative macrophage activation to an M2 "healer" phenotype. Comparative multiplex analysis of MSC- and fibroblast-CdM demonstrated that MSC-CdM contained several factors that may confer therapeutic benefit, including insulin-like growth factor I (IGF-I). Recombinant IGF-I partially reproduced the lung protective effect of MSC-CdM. In summary, MSCs act through a paracrine activity. MSC-CdM promotes the resolution of LPS-induced lung injury by attenuating lung inflammation and promoting a wound healing/anti-inflammatory M2 macrophage phenotype in part via IGF-I.

Cord blood triglycerides are associated with IGF-I levels and contribute to the identification of growth-restricted neonates

ObjectiveThe aim of this study was to investigate whether readily available laboratory tests may aid in the identification of growth-restricted neonates. DesignCord serum levels of 15 chemical analytes, including insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3) were measured in newborns ≥36weeks gestational age (GA). Based on the number of anthropometric indices (out of four) with values ≤25th centile for GA, the babies were allocated into three groups, i.e., Group250, Group251 and Group252 corresponding to neonates with 0, 1 and 2 or more indices, respectively, that were ≤25th centile for GA. Furthermore, two composite variables were developed: A25 (Group250 and Group251) and B25 (Group250 and Group252). The data were evaluated by the Mann–Whitney test and multiple regression analyses. ResultsCord serum triglycerides and total cholesterol levels were significantly higher in Group252 compared to Group250 (p values 0.004 and 0.0009, respectively). The triglycerides almost doubled the power of the variable B25 for predicting IGF-I levels and were found to have a highly significant, negative association with the IGF-I levels (p<0.0001). The IGF-I along with the IGFBP-3 levels explained almost one third of the variation of triglycerides. ConclusionCord serum triglycerides can assist in the identification of growth-restricted neonates. The novel finding of the association of triglycerides with IGF-I calls for further research as this can illuminate unknown aspects of the fetal lipid metabolism.

AKT signaling mediates IGF-I survival actions on otic neural progenitors.

BackgroundOtic neurons and sensory cells derive from common progenitors whose transition into mature cells requires the coordination of cell survival, proliferation and differentiation programmes. Neurotrophic support and survival of post-mitotic otic neurons have been intensively studied, but the bases underlying the regulation of programmed cell death in immature proliferative otic neuroblasts remains poorly understood. The protein kinase AKT acts as a node, playing a critical role in controlling cell survival and cell cycle progression. AKT is activated by trophic factors, including insulin-like growth factor I (IGF-I), through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by phosphatidylinositol 3-kinase (PI3K). Here we have investigated the role of IGF-dependent activation of the PI3K-AKT pathway in maintenance of otic neuroblasts.Methodology/Principal FindingsBy using a combination of organotypic cultures of chicken (Gallus gallus) otic vesicles and acoustic-vestibular ganglia, Western blotting, immunohistochemistry and in situ hybridization, we show that IGF-I-activation of AKT protects neural progenitors from programmed cell death. IGF-I maintains otic neuroblasts in an undifferentiated and proliferative state, which is characterised by the upregulation of the forkhead box M1 (FoxM1) transcription factor. By contrast, our results indicate that post-mitotic p27Kip-positive neurons become IGF-I independent as they extend their neuronal processes. Neurons gradually reduce their expression of the Igf1r, while they increase that of the neurotrophin receptor, TrkC.Conclusions/SignificanceProliferative otic neuroblasts are dependent on the activation of the PI3K-AKT pathway by IGF-I for survival during the otic neuronal progenitor phase of early inner ear development.

IGF-I increases the expression of fibronectin by Nox4-dependent Akt phosphorylation in renal tubular epithelial cells

Extracellular matrix accumulation contributes to the progression of chronic kidney disease. Many growth factors including insulin-like growth factor-I (IGF-I) enhance matrix protein accumulation. Proximal tubular epithelial cells (PTCs) synthesize matrix proteins. NADPH oxidases are major sources of reactive oxygen species (ROS), important signaling molecules that mediate biological responses in a variety of cells and tissue. We investigated the mechanism by which IGF-I regulates fibronectin accumulation in PTCs and the role of a potential redox-dependent signaling pathway. IGF-I induces an increase in NADPH-dependent superoxide generation, enhances the release of hydrogen peroxide, and increases the expression of NADPH oxidase 4 (Nox4) in PTCs. IGF-I also stimulates phosphorylation of Akt, and inhibition of Akt or its upstream activator phosphatidylinositol 3-kinase attenuates IGF-I-induced fibronectin accumulation. Expression of dominant negative Akt also inhibits IGF-I-induced expression of fibronectin, indicating a role for this kinase in fibronectin accumulation. Expression of dominant negative adenovirus Nox4 inhibits IGF-I-induced NADPH oxidase activity, Akt phosphorylation, and fibronectin protein expression. Moreover, transfection of small interfering RNA targeting Nox4 decreases Nox4 protein expression and blocks IGF-I-induced Akt phosphorylation and the increase in fibronectin, placing Nox4 and ROS upstream of Akt signaling pathway. To confirm the role of Nox4, PTCs were infected with adenovirus construct expressing wild-type Nox4. Ad-Nox4, but not control Ad-green fluorescent protein, upregulated Nox4 expression and increased NADPH oxidase activity as well as fibronectin expression. Taken together, these results provide the first evidence for a role of Nox4 in IGF-I-induced Akt phosphorylation and fibronectin expression in tubular epithelial cells.

Expression of Splice Variants of Insulin-Like Growth Factor I and the State of the Myosatellite Cell Pool in the Soleus Muscle in Rats during Gravitational Unweighting and Passive Stretching

Gravitational unweighting is known to induce atrophy and to suppress proliferative processes in the postural muscles; muscle stretching during unweighting allows this atrophy to be overcome. It has been suggested that stretching of the soleus muscle promotes proliferation of satellite cells with subsequent incorporation of their nuclei into fibers on functioning loading of the hindlimbs in rats. The numbers of satellite cells labeled with M-cadherin on cross-sections of single fibers were assessed. After two weeks of antiorthostatic suspension, the number of labeled cells decreased by 33% from the level seen in the control group. In passive stretching, the number of labeled cells was 2.5 times greater than that in suspended animals and 1.7 times greater than in that control animals. The key role in the activation of satellite cells is known to be played by growth factors, including insulin-like growth factor I (IGF-I). Levels of IGF-I expression were measured in the soleus muscle after suspension with stretching, which showed no changes as compared with the suspension without stretching or control groups. Thus, these experiments demonstrated that passive stretching stimulates increases in the proliferation of satellite cells in the soleus muscle of the stretched limb in rats as compared with the level seen in normal conditions, with no changes in the expression of IGF-I or its splice variant MGF.

Is growth hormone expression correlated with variation in growth rate along a latitudinal gradient in Rana temporaria ?

In ectotherms, decreasing season length and lower temperature towards higher latitudes often favour higher growth and developmental rates. However, the underlying physiological mechanisms and particularly the hormonal correlates of clinal variation remain unexplored. The growth hormone (GH) plays a crucial role in growth of all vertebrates and high expression of GH is associated with rapid growth in many species. We tested the hypothesis that GH expression is correlated with a latitudinal gradient in growth in Scandinavian Rana temporaria tadpoles. Using quantitative polymerase chain reaction, we measured GH and growth hormone receptor (GHR) expression at two time points from laboratory-raised tadpoles originating from eight populations collected along the latitudinal gradient. To explore latitudinal differences in stress-induced changes in GH expression, we also compared GH expression in tadpoles raised with and without predators. In accordance with previous studies we found a clear latitudinal gradient in growth. There were no latitudinal effects, or predator-induced effects on GH or GHR expression. However, there was some indication for among-population variation in GH expression. The lack of a latitudinal pattern in GH and GHR expression may be due to that the growth promoting effects of GH is dependent on other factors including insulin-like growth factor-I (IGF), IGF-binding proteins or prolactin. Further studies on these factors may provide insight on the proximal mechanisms of differences in growth in R. temporaria tadpoles.

Apposite Insulin-like Growth Factor (IGF) Receptor Glycosylation Is Critical to the Maintenance of Vascular Smooth Muscle Phenotype in the Presence of Factors Promoting Osteogenic Differentiation and Mineralization

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.

자궁 내 insulin-like growth factor-I 유전자 발현에 미치는 에스트로겐의 영향

자궁은 임신에 필수불가결한 기관으로, 에스트로겐(E2)과 프로게스테론(P4)은 태아와 자궁 사이의 상호 신호전달을 적절하게 조절하여, 임신을 확립하게 하는 필수적 요소이다. 임신 초기 E2는 배아의 안정적인 착상을 위하여 자궁의 성장을 촉진하는 역할을 담당한다. 이 시기 자궁에서 분비되는 인슐린유사 성장인자-I(IGF-I)와 E2/P4 간의 상호 신호전달이 임신 확립에 있어서 매우 중요한 역할을 한다. E2는 에스트로겐 수용체(ER)에 의해 그 작용이 결정되어지는데, 임신 돼지 자궁에서는 ER-<TEX>${\alpha}$</TEX>만이 발현됨을 증명하였다. 자궁에서 ER-<TEX>${\alpha}$</TEX>의 발현은 임신 중기나 말기보다 임신 초기 단계에서 높게 발현됨이 관찰되었다. 이는 배아에서 분비되는 E2가 ER-<TEX>${\alpha}$</TEX>의 발현을 증가시키는 역할을 함을 보여주는 결과이다. IGF-I이 E2 표적 유전자임을 증명하기 위하여 난소를 제거한 쥐에 E2/P4를 처리한 후 IGF-I의 발현을 측정한 결과, E2를 처리한 샘플에서는 IGF-I의 발현이 크게 증가하였으나, P4를 처리한 샘플에서는 큰 영향을 받지 않는 것으로 나타났다. 이 결과는 E2가 자궁에서 IGF-I의 발현을 증가시키는 역할을 수행함을 직접적으로 증명하였다. 임신한 돼지의 자궁에서 임신 시기별로 IGF-I 유전자의 발현을 조사한 결과 임신 초기에 가장 높은 발현을 보였고, 이 양상은 ER-<TEX>${\alpha}$</TEX>의 발현 형태와 아주 유사한 패턴을 나타내었다. 이는 임신 자궁에서 ER-<TEX>${\alpha}$</TEX>와 IGF-I의 발현이 상호 연관되어 있으며 ER-<TEX>${\alpha}$</TEX>와 IGF-I은 E2에 의해서 조절되어지는 유전자이며, 임신초기 배아에서 분비되는 E2가 자궁에서 ER-<TEX>${\alpha}$</TEX>의 발현을 증진시키고, 나아가서 이 ER-<TEX>${\alpha}$</TEX>가 자궁에서 IGF-I 유전자의 발현을 증가시키는 역할을 함을 보여주는 결과이다. The uterus plays a critical role in pregnancy and steroid hormones, and both estrogen (E2) and progesterone (P4) especially play important roles in the cross-talk between embryos and uterus to support the pregnancy. E2 stimulates uterine growth during early pregnancy to prepare for implantation of embryos. This cross-talk during the implantation period involves hormones (E2 and P4) and growth factors, including insulin-like growth factor-I (IGF-I). In the uterus of a pregnant pig, the action of E2 is mediated by estrogen receptor-<TEX>${\beta}$</TEX> (ER-<TEX>${\beta}$</TEX>). The expression of ER-a was much higher in early pregnancy than in mid- and late- pregnancy, suggesting E2 secretion from embryos enhances transcription of ER-a during early pregnancy. In order to prove whether IGF-I is an E2 target gene, quantitative real-time PCR was performed on ovariectomized murine uterus with E2 and/or P4 treatment(s). Increased IGF-I mRNA expression was observed with E2 treatment, however, it was not significantly induced by P4 treatment, which clearly demonstrates that, in mice, E2 depends on the activation of uterine IGF-I gene expression. The expression of IGF-I in the uterus of pigs was much higher in early pregnancy than in mid- and late- pregnancy and these data exhibited the same expression pattern with the ER-<TEX>${\beta}$</TEX> gene expression in the uterus. It suggests that a positive co-relationship between IGF-I and ER-<TEX>${\beta}$</TEX> expression exists in the uterus, and that both gene expressions of IGF-I and ER-<TEX>${\beta}$</TEX> are regulated by E2. It further suggests that uterine the IGF-I gene expression might be initiated by E2 secreted from embryos to increase ER-<TEX>${\beta}$</TEX> gene expression, and that this increased ER-<TEX>${\beta}$</TEX> further stimulates the expression of IGF-I in the uterus during early pregnancy.

Turning anti-ageing genes against cancer

Recent studies in diverse organisms implicate proto-oncogenic pathways, including insulin-like growth factor-I (IGF-I), Ras and AKT/protein kinase B in the ageing process. Although IGF-I is thought to contribute to cancer by promoting growth and preventing apoptosis, evidence from model organisms suggests that proto-oncogene homologues might contribute to the DNA mutations and chromosomal damage that are observed in tumour cells by increasing DNA damage, in both dividing and non-dividing cells, and involving error-prone systems in DNA repair. This raises the possibility that cancer can be reduced by chronic downregulation of pro-ageing pathways.

Reduced susceptibility to two-stage skin carcinogenesis in mice with low circulating insulin-like growth factor I levels.

Calorie restriction has been shown to inhibit epithelial carcinogenesis and this method of dietary restriction reduces many circulating proteins, including insulin-like growth factor I (IGF-I). Previously, we identified a relationship between elevated tissue IGF-I levels and enhanced susceptibility to chemically induced skin tumorigenesis. In this study, liver IGF-I-deficient (LID) mice, which have a 75% reduction in serum IGF-I, were subjected to the standard two-stage skin carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. We observed a significant reduction in epidermal thickness and labeling index in LID mice treated with either vehicle or TPA. A significant decrease in both tumor incidence and tumor multiplicity was observed in LID mice undergoing two-stage skin carcinogenesis relative to wild-type littermates. Western blot analyses of epidermal extracts revealed reduced activation of both the epidermal growth factor and IGF-I receptors in response to TPA treatment in LID mice. In addition, reduced activation of both Akt and the mammalian target of rapamycin (mTOR) was observed in LID mice following TPA treatment relative to wild-type controls. Signaling downstream of mTOR was also reduced. These data suggest a possible mechanism whereby reduced circulating IGF-I leads to attenuated activation of the Akt and mTOR signaling pathways, and thus, diminished epidermal response to tumor promotion, and ultimately, two-stage skin carcinogenesis. The current data also suggest that reduced circulating IGF-I levels which occur as a result of calorie restriction may lead to the inhibition of skin tumorigenesis, at least in part, by a similar mechanism.

CAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation

In the regulation of steroid biosynthesis, a process mediated by the steroidogenic acute regulatory (StAR) protein, both cAMP-dependent and -independent pathways are involved. While the cAMP-dependent regulatory events represent, by far, the most robust increase in steroid synthesis and are well established, the knowledge regarding cAMP-independent mechanisms is lacking. The present investigation was designed to elucidate the potential involvement of the latter in regulating StAR expression and steroidogenesis in mouse Leydig tumor cells (mLTC-1 cells). Treatment of mLTC-1 cells with a number of factors including insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), fibroblast growth factor, transforming growth factor (TGF)alpha, interleukin-1 (IL-1), and colony-stimulating factor-1, increased the levels of StAR mRNA, StAR protein, and progesterone to varying degrees and utilized signaling pathways that are not associated with elevations in intracellular cAMP levels. Importantly, phosphorylation of StAR in response to these stimuli was undetectable, which is in marked contrast to observations with human chorionic gonadotropin (hCG), indicating factors that do not alter intracellular cAMP, regulate the steroid biosynthesis in a StAR phosphorylation-independent manner. In addition, the roles for factors involved in cross-talk between the protein kinase pathways, PKA and PKC, were demonstrated. Further characterization of signaling by one such cAMP-independent factor, TGFalpha, demonstrated that the mechanism, whereby it increased StAR expression and steroid synthesis, was dependent on de novo protein synthesis and mediated via activation of the EGF receptor. TGFalpha was also able to augment hCG-stimulated cAMP synthesis, StAR protein and StAR phosphorylation, and influence hCG binding and LH receptor mRNA expression. Furthermore, TGFalpha increased phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and cAMP-response element-binding protein (CREB), processes inhibited by the mitogen-activated protein kinase/ERK inhibitor U0126 and by expression of non-phosphorylatable CREB-M1 respectively. Inhibition of ERK activity enhanced TGFalpha-mediated StAR protein expression (but not its phosphorylation) and decreased progesterone synthesis, events correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). Collectively, these findings demonstrate that, in mouse Leydig cells, cAMP-independent signaling events regulate steroidogenesis in a StAR phosphorylation-independent manner.

Estrogen-induced changes in IGF-I, Myb family and MAP kinase pathway genes in human uterine leiomyoma and normal uterine smooth muscle cell lines

Many studies have implicated numerous hormones, growth factors, cytokines and other signal transduction molecules in the pathogenesis of uterine leiomyoma. Estrogen and estrogen-related genes are thought to play a key role in the growth of uterine leiomyomas, but the molecular mechanisms are unclear. In an attempt to investigate various pathways that might be involved in estrogen-regulated uterine leiomyoma growth as well as to identify any novel effector genes, microarray studies comparing estrogen-treated uterine leiomyoma cells (UtLM) and normal myometrial cells to untreated cells were performed. Several genes were differentially expressed in estrogen treated UtLM cells, including insulin-like growth factor-I (IGF-I) and others potentially involved in the IGF-I signalling pathway, specifically genes for A-myb, a transcription factor which promotes cell cycle progression and for MKP-1, a dual specificity phosphatase that dephosphorylates mitogen-activated protein kinase. IGF-I and A-myb were up-regulated in estrogen-treated cells while MKP-1 was down-regulated. Two other cell cycle promoting genes, c-fos and myc, were also down-regulated in estrogen treated UtLM cells. These genes are typically up-regulated in response to estrogen in some cells, notably breast epithelial cells, yet consistently have lower expression levels in uterine leiomyoma tissue when compared to autologous myometrium. Our results demonstrate some novel genes that may play a role in the growth of uterine leiomyoma, strengthen the case for involvement of the IGF-I pathway in the response of UtLM to estrogen and corroborate evidence that uterine smooth muscle cells respond to estrogen with a different gene expression pattern than that seen in epithelial cells.

Ras-GRF1 signaling is required for normal beta-cell development and glucose homeostasis.

Development of diabetes generally reflects an inadequate mass of insulin-producing beta-cells. beta-cell proliferation and differentiation are regulated by a variety of growth factors and hormones, including insulin-like growth factor I (IGF-I). GRF1 is a Ras-guanine nucleotide exchange factor known previously for its restricted expression in brain and its role in learning and memory. Here we demonstrate that GRF1 is also expressed in pancreatic islets. Interestingly, our GRF1-deficient mice exhibit reduced body weight, hypoinsulinemia and glucose intolerance owing to a reduction of beta-cells. Whereas insulin resistance is not detected in peripheral tissues, GRF1 knockout mice are leaner due to increased lipid catabolism. The reduction in circulating insulin does not reflect defective glucose sensing or insulin production but results from impaired beta-cell proliferation and reduced neogenesis. IGF-I treatment of isolated islets from GRF1 knockouts fails to activate critical downstream signals such as Akt and Erk. The observed phenotype is similar to manifestations of preclinical type 2 diabetes. Thus, our observations demonstrate a novel and specific role for Ras-GRF1 pathways in the development and maintenance of normal beta-cell number and function.

Mechanisms by which nutritional disorders cause reduced bone mass in adults.

Nutritional disorders that cause bone loss in adults include disordered eating behaviors (female athlete triad and anorexia nervosa), gastrointestinal diseases (celiac sprue, inflammatory bowel disease, and other malabsorption syndromes), alcoholism, and hypervitaminosis A. These disorders exert their effects on bone through a number of mechanisms, including estrogen deficiency. Deficiencies of anabolic hormones may also be important, including insulin-like growth factor I (IGF-I), a nutritionally regulated bone trophic factor. In addition, low weight itself is a risk factor for bone loss and decreased bone formation. Reduced calcium and vitamin D availability, with resultant secondary hyperparathyroidism, is another important mechanism of bone loss in nutritional disorders. This review discusses nutritional causes of reduced bone mass in adults and how nutritional disorders exert deleterious effects on the skeleton.

Effects of insulin-like growth factor-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture under different masticatory loading conditions in growing mice

It is well accepted that mechanical loading inhibits bone resorption and increases in vivo bone formation. It is also known that cyclic mechanical loading, in particular, can enhance bone formation significantly. These findings suggest a significant role for mechanical stimuli in bone remodelling mediated by various local growth factors including insulin-like growth factor-I (IGF-I). Earlier studies showed that the nasal bone length and premaxillary bone width were significantly greater in mice fed a solid diet rather than a granulated diet, and that these dimensions increased significantly in a solid-diet group treated with IGF-I. The present study sought to examine the effect of IGF-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture subjected to different masticatory loadings. For the solid-diet groups, the numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells and osteoblasts were significantly greater in the group injected with IGF-I than in the animals injected with physiological saline. In the groups fed a granulated diet, no significant differences in the numbers of TRAP-positive osteoclastic cells and osteoblasts were found over the entire experimental period between mice injected with either IGF-I or physiological saline. It is shown that IGF-I significantly induces the expression of osteoclasts and osteoblasts and the subsequent bone remodelling, and that the effect may be additive as compared to that of mechanical masticatory loading, which seems to be more important in bone remodelling in terms of the numbers of osteoclasts and osteoblasts.